Sustained increase of intracellular Ca2+ or sustained Ca2+ overload (cytosolic, nuclear and mitochondrial) is known to be associated with many abnormal cell function including hypertension, artherosclerosis, hyperinsulinemia, diabetes Melitus type II, abnormal cell proliferation, cell—cell interactions, necrosis, ischemia/reperfusion, arrythmias, platelet activation and aggregation as well as inflammation and asthma (Bkaily, 1994, Medical Intelligence Unit, CRC Press, Austin; Bkaily and Jacques, 1994, Kluwer Academic Publ., Boston; Bkaily et al., 1994, Kluwer Academic Publ., Boston; Bkaily et al., 1997, Can. J. Physiol. Pharmacol. 75:652–666; Bkaily et al., 1997, Mol. Cell. Biochem. 172:171–194; Bkaily et al., 1997b, Drug Devel. Res. 42:211–222; Sowers et al., 1993, Am. J. Hypert. 6:302–307; 1994; Hurwitz et al., 1991, CRC Press, Boca Raton, Ann Arbor; Nagano et al., 1992, Kluwer Academic Publ., Boston; Anand et al., 1989, Kluwer Academic Publ. Boston; Dhalla et al., 1996, Kluwer Academic Publ. Boston; Karmazyn, 1996, Birkhauser Verlag. Basel, Boston; Curtis, 1993, Academic Press. London, San Diego; De Brum et al., 1996, Br. J. Pharmacol. 118:1597–1604; Foreman, 1993, Academic Press, London, San Diego; Furberg et al., 1993, Am. J. Hypert. 6:24S–29S; Holgate et al., 1993, Academic Press, London, Boston; Jacobs et al., 1993, Hypertension 21:308–314; Johnson et al., 1993, J. Clinic. Pharmacol. 35:484–492; Levy et al., 1994, Am. J. Med. 96:260–273; Raman et al., 1995, Am. J. Hypert. 8:197–200; Sperelakis et al., 1984, Martinus Nijhoff Publ. Boston; Standley et al., 1993; Wray et al., 1989, The New York Academy of Sciences, New York. Vol. 560). A wide variety of drugs has been tested against different types of Ca2+ channels (P, N, T and L) and the development of Ca2+ blockers has been concentrated on the L-type Ca2+ channel, which has never been shown to undergo any abnormal function in many diseases implicating sustained increase of intracellular Ca2+ ([Ca]i) or Ca2+ overload. Also, these drugs with the exception of isradipine (PN200-110, Lomir/Dynacirc), failed to block or prevent the sustained increase of [Ca]i, Ca2+ overload and necrosis. Recently, the presence of a steady-state nifedipine (L-type blocker)-insensitive but isradipine sensitive (dual L and R-type blocker) R-type (resting-type) Ca2+ channel that is voltage and Ligand-G protein-dependent has been reported (Bkaily et al., 1991, Elsevier, New York; Bkaily et al., 1992a, Am. J. Physiol. 262:H463–471; 1993, Br. J. Pharmacol. 110:519–520; 1993a, J. Mol. Cell. Cardiol. 25:1305–1316; 1995, J. Cardiovascul. Pharmacol. 26:303–306; 1996, Mol. Cell. Biochem. 154:113–121; 1997, Drug Develop. Res. 42:211–222; 1997a, Mol. Cell. Biochem. 172:171–194; 1997b, Can. J. Physiol. Pharmacol. 75:652–666; 1997d, Mol. Cell. Biochem. 170:1–8; 1997d, Mol. Cell. Biochem. 176:199–204; 1998, Mol. Cell. Biochem., 183:39–47; Bkaily, 1994a, In: Ionic channels in vascular smooth muscle. G. Bkaily edt. Molecular Biology Intelligence Unit, R.G. Lands Co. Austin.). This channel was responsible for maintaining the resting cytosolic and nuclear Ca2+ levels and its overstimulation by sustained depolarization or by permanent presence of some hormones such as insulin, ET-1, PAF, TNFα, PDGF, Bradykinin, or IL-1 induced sustained increase of [Ca]c and [Ca]n. (Bkaily et al., 1991, Elsevier, New York; 1993, supra; 1995, supra; 1996, supra, 1997a, supra; 1997b, supra; 1997c, supra; 1997d, supra; 1998, supra; Bkaily, 1994a, supra; Bkaily, 1994b, In: Membrane physiopathology. G. Bkaily edt. Kluwer Acad. Publ. Boston; Taoudi et al., 1995, J. Cardiovasc Pharmacol. 26:300–302). The important features that distinguish this channel from other Ca2+ channels are the sustained activity (as long as a depolarization or the pharmacological and physiological agonist is present) and the large number of disparate agonists that indirectly (via receptor-G proteins coupling) stimulate the channel.
Several reviews described the presence of various types of voltage-dependent Ca2+ channels in many cell types including heart, vascular smooth muscle (VSM) and vascular endothelial (VE) cells (Godfraind and Govoni, 1995; Bkaily, 1994b, supra; Orallo, 1996, Bkaily et al., 1997a, supra). Among these different types of Ca2+ channels, the resting membrane potential steady-state voltage-dependent R-type (for resting) Ca2+ channel was first reported by the group of Bkaily et al. (Bkaily et al., 1991, supra; 1992, supra; Bkaily, 1994, supra). Later, the group of Tsien (Zhang et al., 1993, Neuropharmacol 32:1075–1088; Randall et al., 1997, Neuropharmacol 36:879–893) described a dihydropyridine resistant type Ca2+ channel also named R-type (for resistant).
The steady-state R-type Ca2+ channel in human VSM and VE cells was reported to possess a nearly 24 pS single channel conductance (in 110 mM Ca2+) (Bkaily et al., 1997a, supra). This type of channel was shown to be responsible for determining, under normal conditions, the resting tension of VSM cells and secretions by VE cells (Bkaily et al., 1991, supra; Bkaily et al., 1992, supra; Bkaily et al., 1993, supra; Bkaily et al., 1995, supra; Bkaily et al., 1996; Bkaily et al., 1997a; Bkaily et al., 1997b; Claing et al., 1994, Br. J. Pharmacol. 1:1202–1208; Taoudi-Benchekroun et al., 1995, J. Cardiovasc. Pharmacol. 26:300–302). This type of Ca2+ channel is known to be insensitive to nifedipine and inorganic L-type Ca2+ channel blockers such as cobalt, cadmium and Mn2+ and the T-type Ca2+ blocker, nickel (Bkaily, 1994, supra). However, it is blocked by PN200-110 (isradipine) which is also known to block the L-type Ca2+ channel (Bkaily et al., 1992, supra; Bkaily et al., 1997a, supra). Unlike T and L-type Ca2+ channels, the R-type Ca2+ channel was not regulated by second messengers such as cAMP, cGMP and protein kinase C and neither by ATP (Bkaily, 1994, supra). This type of channel was reported to be indirectly stimulated by insulin, PAF, ET-1 and bradykinin via stimulation of a PTX and CTX sensitive G-protein(s) (Bkaily, 1994a, supra; 1991, supra; 1992, supra; 1995; 1996, supra; 1997a, supra; 1997b, supra; 1997c, supra; 1998, supra) and to contribute to a sustained elevation of cytosolic ([Ca]c) and nuclear ([Ca]n) Ca2+. These indirect R-type Ca2+ channel stimulators such as PAF induced elevation of [Ca]c and [Ca]n by increasing the probability of opening of the channel, and thus allowing longer influx of Ca2+ through the sarcolemmal membrane (Bkaily et al., 1997a, supra).
Since PN200-110 was found to be the only available compound to depress the R-type Ca2+ channel and since this Ca2+ blocker is known to affect other types of Ca2+ channels, there remains a need to develop specific and potent steady-state R-type Ca2+ channel blockers.
The steady-state R-type Ca2+ channels are distributed in a non-homogenous fashion, similarly to some other receptors. This type of channel seems to have no inactivation gate and it is highly selective for Ca2+ ions. The R-type Ca2+ channel is highly voltage-dependent but could be stimulated by receptors whose activation is coupled to a specific PTX and CTX-sensitive G-protein(s) (Bkaily et al., 1998, supra). Thus, if the R-type Ca2+ channel is fully activated via a receptor dependent pathway, it may appear as a receptor operated Ca2+ channel. Moreover, if the R-type channel is fully activated by voltage, receptor stimulation does not further modulate its function and appears as a pure voltage-dependent channel (Bkaily, 1994, supra; 1997a, supra; 1997c, supra). Since T- and L-type Ca2+ channels are rapidly inactivated during sustained voltage or pharmacological stimulation, these types of channels can only contribute to the inset stimulation. However, the R-type Ca2+ channel will contribute to both inset and sustained elevation of cytosolic and nuclear free Ca2+, seen in normal and pathological conditions, depending on the function of the studied cell type. Hence, this type of channel, under normal physiological situations contributes to the resting Ca2+ influx responsible for determining the resting cytosolic and nuclear Ca2+ that modulate resting tension, secretion, protein synthesis and mitosis. In working muscle cells, such as heart cells, the normal physiological function of this channel at the sarcolemmal membrane level, is to maintain normal resting cytosolic Ca2+ level. However, at the nuclear membrane levels, this channel seems to be implicated in maintaining normal resting nucleoplasmic Ca2+ levels (near 300 nM) (Bkaily et al., 1997a, supra; 1997b, supra).
During excitation-contraction coupling, the R-type Ca2+ channel is implicated in regulating Ca2+ wave propagation initiated, by Ca2+ influx through the opening of the T- and L-type Ca2+ channels and the subsequent large Ca2+ release from the SR by attenuating the cytosolic Ca2+ wave amplitude, by allowing Ca2+ influx through the nuclear membrane and thus permitting a smooth contraction and relaxation. The subsequent release of the uptaken Ca2+ permits the maintainance of Ca2+ waves and slow relaxation and propagation of the waves to neighboring cells, most likely through gap-junctions and in this manner, allowing synchronization of contraction of ventricular cells (Lopez et al., 1995, Biochem Biophys Res Commun 214:781–787; Bkaily et al., 1996, supra; 1997a, supra). The fact that cytosolic Ca2+ waves cannot be completely absorbed by the nucleus is due to the maximum Ca2+ buffering capacity of the nucleus which is shielded from variations in cytoplasmic Ca2+, perhaps by gating mechanisms in the perinuclear envelope once its maximum capacity is reached (Burnier et al., 1994, Am J Physiol 266:C1118–C1127; Bkaily, 1994, supra; 1996, supra; 1997a, supra, 1997b, supra).
Recent published results also showed that in secretory cells such as VE and VSM cells, tonic secretion or contraction is mainly, if not only due to the activation of sarcolemmal R-type Ca2+ channels. It was further shown that in VSM and excitable cells (VE cells do not possess T- or L-type Ca2+ channels) the T and/or the L-type Ca2+ channel activation, serves as a turbo Ca2+ influx mechanism in order to rapidly bring the Ca2+ level up to the threshold level for contractile elements and to pre-overload the nucleoplasm with Ca2+, enabling cytosolic accumulation of Ca2+ and maintain tension. Thus, overstimulation of the R-type Ca2+ channels may highly contribute to cytosolic and nuclear Ca2+ accumulation that could be considered in many cases as the first and in all cases the final pathological consequence of several diseases such as hypertension, atherosclerosis, abnormal conduction, arythmias, fibrillation, and of remodelling, proliferation and apoptosis. For these reasons, targeting the sarcolemmal nuclear membrane R-type Ca2+ channels with a selective depressor blocker, or targeting receptors that indirectly modulate this type of channel at the sarcolemmal, or mainly at the nuclear membrane level, would constitute without any doubt, a major therapeutical pathway for a new generation of Ca2+ channel and Ca2+ entry blockers.
For example, the sustained activation of the R-type channel by insulin may explain in part “syndrome X”, the hypertension, hyperglycemia, dyslipidemia, vascular smooth muscle proliferation and end organ damage associated with non-insulin-dependent diabetes mellitus (NIDDM) and obesity-induced hypertension. Also, the sustained increase in [Ca]c and mainly [Ca]n mediated by the stimulation of the R-type Ca2+ channel could contribute to the expression of oncogenes and to the proliferation of malignant cells as well as to stimulation of TNFα; PAF which would lead to septic shock. The finding that Lomir/Dynacirc (but none of the other L-type Ca2+ antagonists) is unique in depressing the overstimulation of the R-type Ca2+ channel permits the identification and characterization of this type of Ca2+ channel. Non published results in two human osteoblast cancer lines (MG63 and FAOS-2) clearly showed that Lomir/Dynacirc (10−8M) reduced spontaneous cell proliferation and blocked hypertension and ET-1 plasma elevation associated with cyclosporin A treatment in allograft transplant. In contrast, an L-type Ca2+ blocker, nifedipine (10−6M) had no effect. A role for the R-type Ca2+ channel and Lomir/Dynacirc in human cancer is suggested by the above findings and supported by the finding of reduced cancer rates in the Lomir/Dynacirc treated group of the MIDAS study. The identification of a potent and specific antagonists may hold the possibility of a new therapeutic target for novel medications. The novel R-type Ca2+ channel may also prove important in dissecting differential signalling pathways in immune cells. The evaluation of these mechanisms leads to R-type blockade as a therapeutic tool for specific intervention in graft rejection, autoimmune diseases, asthma and septic shock.
A recent report in patients with type I and type II Raynaud's phenomenon (pain and numbness in the fingers, which in some subjects can be complicated by skin ulcers) showed that Lomir/Dynacirc significantly reduced the elevated plasma concentration of ET-1 level, frequency, severity, and disabling nature of acute attacks of Raynaud's phenomenon (La Civita et al., 1996, Clinic. Drugs Invest 11:S126–31). The decrease of the elevated ET-1 circulating level by Lomir/Dynacirc is due to the blockade of the R-type Ca2+ channel which reverses the sustained increase of [Ca]c and [Ca]n, thus, reducing the autocrine and self perpetuating secretion of mitogenic factors such as ET-1, PAF and TNFα. A blockade of the elevated autocrine and self perpetuating secretion of mitogenic factors by cancer cells may in turn contribute to reduction and even blockade of expression of oncogenes and proliferation of these cells.
The use of Sandimmune is known to produce potentially serious side effects such as renal impairment and hypertension. These side effects will restrict Sandiummune's use in autoimmune indications such as psoriasis and rheumatoid arthritis. The renal impairment and hypertension are attributable to altered renal hemodynamics induced by Sandimmune. The L-type Ca2+-channel blockers have been used successfully to treat hypertension and renal impairment. The benefits of the Ca2+-channel blockers have been attributed to their effects on renal hemodynamics specifically dilation of the afferent renal arteriole.
Data indicate that the dual R- and L-type Ca2+ channel blocker isradipine (but not a pure L-type blocker) may correct the vasoconstriction at both the afferent and efferent renal arterioles. The advantage of dilation of the afferent and efferent arterioles is a correction of renal blood flow and glomerular filtration without an increase in filtration fraction. Filtration fraction is an indicator of filtration pressure. An increase in filtration pressure could increase the likelihood of developing glomerulonephritis and eventual renal failure.
The potential benefit of blockade of the R-type Ca2+ channel by Lomir/Dynacirc on filtration pressure is supported by the existing literature. For example Grossman et al. (1991, Am. J. Cardiol. 68:65–70) in a 3-month study with Lomir/Dynacirc showed that filtration fraction remained constant. The filtration fraction remained constant despite the increase in glomerular filtration rate and renal blood flow. Vascular resistance was also reduced by the 3 month treatment with Lomir/Dynacirc.
A favourable effect on filtration fraction has been corroborated in transplant patients (Berg et al., 1991, Nephrology, Dialysis, Transplantation. 6:725–30). These investigators showed that filtration fraction was reduced by Lomir/Dynacirc while renal blood flow increased.
The R-type Ca2+ channel has also been identified and characterised in vascular smooth muscle cells isolated from human renal arteries (Bkaily et al., 1991, supra).
Mandevilla velutina is a native Brazilian plant used in folk medicine to treat snake bites and as an anti-inflammatory agent. Some non-peptidic compounds extracted from this plant block bradykinin and related kinins action. It shows potent analgesic and anti-inflammatory activities (Calixto et al., 1987, supra).
Since 1985, Calixto's group has worked on extracts of Mandevilla velutina (MV) and claimed that some of the extracts (such as MV8608) had antagonistic properties against the effect of bradykinin (BK). The compound MV8608 has been characterized in 1987 (Calixto et al., Br. J. Pharmacol. 91:199–204). It has been found to be selective in its ability to inhibit the contraction of rat uterus induced by BK. The previous work made by Calixto's group as well as others on extracts of Mandevilla species have always focused on compounds which have a presumed action at the BK receptor site.
In a review article published after 1990, Calixto's group (Calixto and Yunes, 1991, Mem. Inst. Oswaldo 86:195–202, supl. 2) mentioned that the compounds MV8608 had a pregnane structure. It is further mentioned that MV8608 is an aglycone compound (without any sugar). No specific structure is shown in this review article concerning MV8612. This review is a compendium of data, characteristics, and properties of MV8608 and MV8612 in numerous systems responding to BK (therefore not limited to the effect of BK on rat uterus). Again, it may be deducted from this publication that the Calixto group of researchers have focused their study on the search of a ligand which is a BK receptor antagonist. MV8612 has been retained as a good candidate because it best corresponds to established receptor classification criteria (a fairly good pA2, competition curve whose slope does not differ from one and selectivity). Of note, Calixto publication (Calixto and Yunes, 1991, supra) does not teach or suggest that MV8612 may have an action which is aimed at the receptor directly. Although on certain systems the effect of MV8612 has been shown to be non-selective, no explanation on this lack of selectivity toward BK has been provided. Therefore, this publication does not teach or suggest any role of MV8608 and MV8612 as calcium channel blockers.
Other compounds isolated from Mandevilla Pentlandia, have also claimed an anti-BK activity, (patent application of Proctor and Gamble Co., EP 0/359310). Furthermore, other Mandevilla extracts, particularly from Mandevilla Illustris have been shown to have physiological antagonist activity against BK. Indeed, all the compounds obtained from Mandevilla species are described as compounds having anti-BK activity. Strikingly, all such descriptions fail to teach or mention the specific site of action of these compounds, and while they lack selectivity, they are deemed to be useful for treating pathologies and conditions involving bradykinin (inflammation, smooth muscle contraction, pain, hypotension, etc.).
The art teaches that a non-specific inhibitor of a calcium channel such as isradipine, which has an effect on calcium channel types L and R, reduces or abolishes the effect of hormones like insulin and PAF (platelet-activating factor), ET-1 and BK which effect is absent when using nifedipine (a L-channel blocker). Nevertheless, the art is indicative of the contribution of the R-type calcium channel in the effect of insulin, PAF, ET-1 and BK.
There thus remains a need to assess the specificity of MV8608 and MV8612 by identifying their direct or indirect effects on Ca2+ homeostasis. More broadly, there remains a need to verify whether MV8608 and MV8612 are as non-specific as isradipine. More particularly, there remains a need to assess the activity of these compounds on the R-type Ca2+ channel, as well as T, L Ca2+ channels and the fast Na+ and delayed outward K+ channels.
In spite of the recent discovery of the R-type Ca2+ channel, there is a definite need for a new generation of class of drugs to treat overstimulation of R-type Ca2+ channel-associated diseases for the following reasons:
1. There is no drug approved for the treatment of diseases or conditions in which a sustained elevation of [Ca]c, [Ca]n or R-type Ca2+ blocking is encountered; and
2. There remains a definite need for the identification of drugs which are more specific, show less side effects and have a wider therapeutic value, for the treatment of hypertension, artherosclerosis, inflammation, septic shock, arthritis, asthma, cancer, pain, diabetes type II and ischemia-reperfusion, hyperventilation and high circulating ET-1 level.
The present invention seeks to meet these and other needs.